Immunoassays for Lipid Peroxidation End Products

One-Hour ELISA for Protein-Bound Acrolein and HNE
  • Kimihiko Satoh
  • Koji Uchida
Part of the Methods in Pharmacology and Toxicology book series (MIPT)


Unsaturated aldehydes such as acrolein and hydroxyalkenals are produced in vivo through lipid peroxidation chain reactions under conditions of oxidative stress or carcinogenic insult, and are causally involved in the pathogenesis of numerous diseases (1,2). The ability of cytotoxic and genotoxic compounds to inactivate various biologically important macromolecules has been well documented (3, 4, 5). For enzymes and proteins, lysine, cysteine, histidine, and other amino acid residues that play key roles in their functionings are preferentially modified. The complicated chemistry and biochemistry of the reactive molecules occurring in vivo in micro- or ultramicroquantities remain unclear, because direct determination of such unstable compounds is problematic. An alternative approach is the immunochemical quantification of reaction products accumulating in vivo, for which monoclonal antibodies (MAbs) specific for the acrolein-modified lysine and 4-hydroxy-2-nonenal (HNE)-modified histidine epitopes have been prepared (Fig. 1) (6,7). However, several difficulties are encountered with enzyme-linked immunosorbent assay (ELISA) determination of haptenic molecules. One is the low avidity of MAbs against the small epitopes in general. The other is that the most widely used sandwich method is unapplicable to the quantitation because monovalent antigens are undetectable in principle.
Fig. 1.

Structures of acrolein and HNE (A) and their amino acid adducts (B).


Antigen Coating Sandwich Method Genotoxic Compound Minimal Reaction Time Important Macromolecule 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Hallwell, B. and Gutteridge, J. M. C. (1998) Free Radicals in Biology and Medicine, 3rd ed. Oxford Science Publ. Great Clarendon.Google Scholar
  2. 2.
    Esterbauer, H., Schaur, R. J., and Zollner, H. (1991) Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic. Biol. Med. 11, 81–128.PubMedCrossRefGoogle Scholar
  3. 3.
    Benedetti, A., Comporti, M., and Esterbauer, H. (1980) Identification of 4-hydroxynonenal as a cytotoxic product originating from the peroxidation of liver microsomal lipids. Biochim. Biophys. Acta 620, 281–296.PubMedGoogle Scholar
  4. 4.
    Uchida, K. and Stadtman, E. R. (1992) Modification of histidine residues in proteins by reaction with 4-hydroxynonenal. Proc. Natl. Acad Sci. USA 89, 4544–4548.PubMedCrossRefGoogle Scholar
  5. 5.
    Satoh, K., Kitahara, A., Soma, Y., Inaba, Y., Hatayama, I., and Sato, K. (1985) Purification, induction, and distribution of placental glutathione transferase: a new marker enzyme for preneoplastic cells in the rat chemical hepatocarcinogenesis. Proc. Natl. Acad. Sci. USA 82, 3964–3968.PubMedCrossRefGoogle Scholar
  6. 6.
    Toyokuni, S., Miyake, N., Hiai, H., Hagiwara, M., Kawakishi, S., Osawa, T., and Uchida, K. (1995) The monoclonal antibody specific for the 4-hydroxy-2-nonenal histidine adduct. FEBS Lett. 359, 189–191.PubMedCrossRefGoogle Scholar
  7. 7.
    Uchida, K., Kanematsu, M., Sakai, K., et al. (1998) Protein-bound acrolein: potential markers for oxidative stress. Proc. Natl. Acad. Sci. USA 95, 4852–4887.Google Scholar
  8. 8.
    Satoh, K., Yamada, S., Koike, Y., et al. (1999) A 1-hour enzyme-linked immunosorbent assay for quantitation of acrolein-and hydroxynonenal-modified proteins by epitope-bound casein matrix method. Anal. Biochem. 270, 323–328.PubMedCrossRefGoogle Scholar
  9. 9.
    Uchida, K., Kanematsu, M., Morimitsu, Y., Osawa, T., Noguchi, N., and Niki, E. (1998) Acrolein is a product of lipid peroxidation reaction. Formation of free acrolein and its conjugate with lysine residues in oxidized low density lipoproteins. J. Biol. Chem. 273, 16,058–16,066.PubMedCrossRefGoogle Scholar
  10. 10.
    Duhamel, R. C. and Johnson, D. A. (1985) Use of nonfat dry milk to block nonspecific nuclear and membrane staining by avidin conjugates. J. Histochem. Cytochem. 33, 711–714.PubMedGoogle Scholar
  11. 11.
    Froese, A., Sehon, A. H., and Eigen, M. (1962) Kinetic studies of protein-dye and antibody-hapten interactions with the temperature-jump method. Canad. J. Chem. 40, 1786–1797.CrossRefGoogle Scholar
  12. 12.
    Day, L. A., Sturtevant, J. M., and Singer, S. J. (1962) The kinetics of the reactions between antibodies to the 2,4 dinitrophenyl group and specific haptens. Ann. N. Y. Acad. Sci. 103, 611–625.CrossRefGoogle Scholar
  13. 13.
    Kabat, E. A. and Mayer, N. M. (1961) Experimental immunochemistry, 2nd ed., Charles C. Thomas Publ., Springfield, IL, pp. 22–96.Google Scholar

Copyright information

© Humana Press Inc.,Totowa, NJ 2003

Authors and Affiliations

  • Kimihiko Satoh
    • 1
  • Koji Uchida
    • 2
  1. 1.Department of Organic FunctionHirosaki University School of Health SciencesHirosakiJapan
  2. 2.Laboratory of Food and Biodynamics, Graduate School of Bioagricultural SciencesNagoya UniversityNagoyaJapan

Personalised recommendations